Flow cytometry (FCM) is a versatile multicolor technology for multi-parameter analysis which is unparalleled in its facility and capacity for bioassay multiplexing, the performance of multiple assays in a single sample volume. By increasing multi-sample throughput 20-fold, recently developed HyperCyt technology has overcome historic barriers to application of this and other powerful features of FCM in the high throughput screening (HTS) drug and probe discovery enterprise. Specific aims here address the long term goal of a robust FCM platform capable of extended, fully automated operation in the HTS environment. In Aim 1 the HyperCyt platform will be modified to process four 384-well quadrants of a 1536-well plate in parallel, using four sampling probes to send samples to four separate flow cytometers by positive pressure sample delivery. This approach will take advantage of powerful, low-cost flow cytometers to quadruple throughput to about 8,400 samples/hour. Aim 2 will evaluate an alternative approach in which samples will be pulled rather than pushed through the cytometer observation chamber. Exploiting a cytometer that uses negative pressure for sample intake, an automated XYZ stage will introduce air-bubble separated bioassay samples directly into the cytometer from 1536-well plates without need of the HyperCyt peristaltic pump. This will eliminate cell exposure to compressive pump forces, shorten sample transit distance to reduce fluid carryover between samples, and allow sample fluid to be moved at higher velocity without loss of optical resolution to achieve an expected doubling of throughput for a single cytometer. Aim 3 will beta-test each platform in the University of New Mexico Center for Molecular Discovery (UNMCMD). Established multiplexed assays will be used to optimize and validate platform performance vs. mature 384 well FCM HTS technology. Samples containing 20 or more single-plex elements (i.e., e 20-plex) will be used to define the limits of and balance between multiplex composition and sample throughput. By the end of the project period, these novel platforms will be positioned for integration into the automated process stream of the UNMCMD, allowing HTS FCM to take its place in the armamentarium of robust HTS capabilities for the Molecular Libraries Probe Centers Network and for the international scientific community. PUBLIC HEALTH RELEVANCE: Flow cytometry has proven to be a pivotal technology in biological investigations of health and disease (AIDS, Genome Project, stem cells, autoimmune disease, and cancer, to name a few). Two proposed innovative platforms promise complementary solutions by which to significantly increase the speed and efficiency of flow cytometry in small molecule high throughput screening, advancing discovery of novel drugs and biological probes.